ERCC 1 isoform expression and DNA repair in NSCLC

NEJM 2013;368:1101Reporter: 胡名宏

Supervisor: 邱宗傑 2013.06.03

Predictive/Prognostic factors in NSCLC

Predictive Prognostic

EGFR Benefit from EGFR TKI

KRAS Lack benefit from platinum/vinorelbine or EGFR TKI

Poor survival

ALK fusion gene Benefit from ALK inhibitor

High ERCC1 expression

Lack benefit from platinum –based chemotherapy

Better survival

IALT

RANDOIZE

Cisplatin-based adjuvant C/T (n=935)

Observation (n=932)

N=1867Stage I~IIINSCLCCompleted resectedOptional RT

R Primary: OS

NEJM 2004;350:351JCO 2010;28:35

IALT

NEJM 2004;350:351

5yr OS 44.5% vs 40.4% (P<0.03) 5yr RFS 39.4% vs 34.3% (P<0.003)

JBR.10

RANDOIZE

CDDP+Vinorelbine adjuvant C/T (n=242)

Observation (n=240)

N=482Stage IB, IINSCLCCompleted resectedAge ≥18PS 0-1

R Primary: OS

NEJM 2005;352:2589JCO 2010;28:29

JBR.10

NEJM 2005;352:2589

5-yr RFS rate 39.4% vs 34.3% (P<0.001)

5-yr OS rate 69% vs 54% (P=0.009)

Excision repair cross-complementation group 1 (ERCC1)

Ribonucleotide reductase M1 (RRM1)

ERCC-1 staining

NEJM 2007;356:800

IALT

NEJM 2006;305:983

ERCC negative tumorOS HR 0.65 (0.50~0.86) (P=0.002)

ERCC negative tumorDFS HR 0.65 (0.50~0.85) (P=0.001)

IALT

ERCC positive tumorOS HR 1.40 (0.84~1.55) (P=0.40)

NEJM 2006;305:983

DFS and OS for high RRM1/ERCC1 NSCLC

NEJM 2007;356:800

DFS > 120m in highRRM1/high ERCC1 (P=0.01)

OS > 120m in highRRM1/high ERCC1 (P=0.02)

Background

• DNA repair capacity is a major determinant of cisplatin resistance

• ERCC1 protein plays an essential role in nucleotide excision repair.

• ERCC1 as a biomarker of patient survival, treatment efficacy, or both has been studied at the genomic level, transcriptional level and protein level in both retrospective and prospective studies.

Background

• The ERCC1 gene generates four isoforms (designated 201, 202, 203, and 204) by alternative splicing

• ERCC1-201 and ERCC1-203 isoform have appeared to be nonfunctional in nucleotide excision repair capacity

• Previous study revealed level of expression of ERCC1 in NSCLC tumors was prognostic or predictive, or both, of a benefit from cisplatin-based adjuvant chemotherapy

Method

• Tumor samples from the IALT, Cancer and Leukemia Group B (CALGB) 9633, and National Cancer Institute of Canada Clinical Trials Group JBR.10 trials are included in the LACE Biology biomarker project.

• GALGB 9633 (180 P’t) and JBR.10 (314 P’t): validation set

• 589 P’t from IALT could be stained again

Method

• Mouse monoclonal antibody against ERCC1 (clone 8F1) were used

• Both set were evaluated by experienced pathologist in a blinded fashion

• Stroma, epitheliuim and endothelium cells were taken into account

• Staining intensity scale 0~3 (percentage of positive tumor nuclei 0% for 0, 0~9% for 0.1, 10~49% for 0.5, >50% for 1.0)

• ERCC H score>1 was ERCC1-positive

A549 cell line

• ERCC1-deficient cells after knocked out ERCC1 gene

• High sensitivity to cisplatin and a low rate of repair of cisplatin–DNA adducts

Results

• ERCC1 was scored as positive (H score >1) in 78% of samples (494 patients in the validation set)

• Among patients with ERCC1-negative or ERCC-positive tumors, overall survival did not differ significantly between the chemotherapy and control groups.

OS in ERCC-neg and ERCC-pos with chemotherapy

HR 1.16; 95% [CI] 0.64 to 2.10(P = 0.62)

HR 0.78; 95% [CI] 0.58 to 1.05( P = 0.09)

Results

• Discrepancy of ERCC1 tumor staining between 2006 and 2011 was noted:

ERCC1 (+) only 44% of the IALT Biology cohort in 2006, but , 77% were scored as positive using current 8F1 antibody batch

Discrepancy of ERCC1 tumor staining

• Discordant sample 36%• Possible change in 8F1

antibody batch might increase sensitivity

Results

• In addition to 8F1 and FL-297, 14 other commercial Ab used for detecting ERCC

• None of the 16 Abs was specific for only one ERCC1 isoforms

• Using RT-PCR and Western blot, 4 isoforms of ERCC1 could not recognized specifically

Mapping ERCC Abs across different isoforms

Highly immunogenic region

Quantification of removal of cisplatin-DNA adducts

• A459: wild type• ERCC1 –deficient

clone 216 and 375 (control vector)

• Cells expressing single isoforms

• 2-hr cisplatin treatment (25umol/L)

Tumor volumes after treating ERCC1-deficient cell

• 105 ERCC1-deficient cell with single isoforms expression (201, 202,203,204)

• Nude mice• Twice weekly IP

cisplatin infection

IC 50 of cisplatin

• All cell line treated for 48 hrs with increasing dose of cisplatin

• Significant differen -ces from wild type cell (P<0.05)

Discussion

• A number of clinical studies suggest ERCC1 was a prognostic factors or a predictive biomarkers.

• In this study, ERCC1 failed to correlate with overall survival.

Possible explanations

• The current tools used to evaluate ERCC1 expression are inadequate

differences between two 8F1 batches could be related to distinct Ab titration, affinity, purity or even epitope recognition

Possible explanations

• The level of biologic complexity has been underestimated: four ERCC1 protein isoforms have not been correctly assessed

strong homology among the four protein isoforms

nonfunctional isoforms lead to a false classification as ERCC1-positive

ERCC1-202

• Only the reintroduction of the ERCC1-202 isoform rescued nucleotide excision repair activity and the capacity to repair cisplatin-induced DNA damage.

• The unique functional isoform ERCC1-202 might a more accurate predictor marker

Thanks for the listening

Discussion and Comments